Compositions and methods for identification of a duplicate sequencing read

ABSTRACT

The present invention provides methods, compositions and kits for detecting duplicate sequencing reads. In some embodiments, the duplicate sequencing reads are removed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/903,826, filed Nov. 13, 2013, which is herein incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:NUGN_001_01US_SeqList_ST25.txt, date recorded: Nov. 12, 2014, file size3 kilobytes).

FIELD OF THE PRESENT INVENTION

The present invention relates generally to the field of high throughputsequencing reactions and the ability to discern artifacts arisingthrough sequence duplications from nucleotide molecules that are uniquemolecules.

BACKGROUND OF THE PRESENT INVENTION

In RNA sequencing applications, accurate gene expression measurementsmay be hampered by PCR duplicate artifacts that occur during libraryamplification. When analyzing RNA sequencing data, when two or moreidentical sequences are found, it can be difficult to know if theserepresent unique cDNA molecules derived independently from different RNAmolecules, or if they are PCR duplicates derived from a single RNAmolecule. In genotyping by sequencing, duplicate reads can be considerednon-informative and may be collapsed down to a single read, thusreducing the number of sequencing reads used in final analysis.Generally, sequencing reads may be determined to be duplicates if bothforward and reverse reads have identical starting positions, even thoughtwo independently generated molecules can have identical startingpositions by random chance. Single primer extension based targetedresequencing suffers from an issue in that only one end of a sequencingread is randomly generated, while the other (reverse read) end isgenerated by a specific probe. This may make it difficult to determineif two reads are duplicates because they have been duplicated by PCR orbecause by chance they happened to start at the same position.

In expression analysis studies there may be limited value in doingpaired end sequencing since the goal of the experiment is to determineamounts of transcript present as opposed to studying exon usage. Inthese studies, paired end sequencing adds costs while the only value isin helping distinguish PCR duplicates. The probability of two readsstarting in the same position on only one end is higher than theprobability of two reads having the same starting position on two ends(forward and reverse read). There is a need for improved methods thatallow for low-cost, high throughput sequencing of regions of interest,genotyping or simple detection of RNA transcripts without inherentinstrument inefficiencies that drive up sequencing costs due to thegeneration of unusable or non-desired data reads. The inventiondescribed herein fulfills this need. Here, we describe an adaptorapproach that allows for the identification of true PCR duplicates andtheir removal.

The methods of the present invention provide novel methods foridentifying true duplicate reads during sequencing, such as to improvedata analysis of sequencing data, and other related advantages.

SUMMARY OF THE PRESENT INVENTION

The present invention is based, in part, on compositions and methods fordiscerning duplicate sequencing reads from a population of sequencingreads. The detection and/or removal of duplicate sequencing readspresented herein is a novel approach to increasing the efficacy ofevaluating data generated from high throughput sequence reactions,including complex multiplex sequence reactions.

Accordingly, the present invention provides a method of detecting aduplicate sequencing read from a population of sample sequencing reads,the method comprising ligating an adaptor to a 5′ end of each nucleicacid fragment of a plurality of nucleic acid fragments from one or moresamples, wherein the adaptor comprises an indexing primer binding site,an indexing site, an identifier site, and a target sequence primerbinding site. The ligated adaptor-nucleic acid fragment products can beamplified, thus generating a population of sequencing reads from theamplified adaptor-nucleic acid ligation products. The sequencing readswith a duplicate identifier site and target sequence can then bedetected from the population of sequencing reads. The methods canfurther include the removal of the sequencing reads with the duplicateidentifier site and target sequence from the population of sequencereads.

In some embodiments, the identifier site is sequenced with the indexingsite or the target sequence. In further embodiments, the identifier siteis sequenced separately from the indexing site or the target sequence.

In some embodiments, the adaptor comprises from 5′ to 3′ the indexingprimer binding site; the indexing site; the identifier site; and thetarget sequence primer binding site. In further embodiments, the adaptorcomprises from 5′ to 3′ the indexing primer binding site; the indexingsite; the target sequence primer binding site; and the identifier site.

In some embodiments, the plurality of nucleic acid fragments isgenerated from more than one sample. In some embodiments, the nucleicacid fragments from each sample have the same indexing site. In someembodiments, the sequencing reads are separated based on the indexingsite. In yet other embodiments, the separation of sequencing reads isperformed prior to detecting sequence reads with a duplicate identifiersite and target sequence.

In some embodiments, the nucleic acid fragments are DNA fragments, RNAfragments, or DNA/RNA fragments. In further embodiments, the nucleicacid fragments are genomic DNA fragments or cDNA fragments.

In some embodiments, the indexing site is between 2 and 8 nucleotides inlength. In further embodiments, the indexing site is about 6 nucleotidesin length. In some embodiments, the identifier site is between 1 and 8nucleotides in length. In further embodiments, the identifier site isabout 8 nucleotides in length.

In some embodiments, the indexing primer binding site is a universalindexing primer binding site; and in some embodiments, the targetsequence primer binding site is a universal target sequence primerbinding site.

The present invention also encompasses embodiments that include a kitcomprising a plurality of adaptors, wherein each adaptor comprise anindexing primer binding site; an indexing site, and identifier site, anda target sequencing primer binding site.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the novel features of the invention andadvantages of the present invention will be obtained by reference to thefollowing description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1 depicts a schematic of generating sequencing reads of a libraryincluding where an indexing primer and a target sequence primer anneal.

FIG. 2A depicts a mechanism of the Single Primer Enrichment Technology(SPET) and how an identifier site is carried over into the final libraryand how sequencing through the indexing site into the identifier siteprovides data on which nucleic acid molecule is being identified.

FIG. 2B is a continuation of FIG. 2A.

FIG. 3 offers a detailed view of the sequence of an example adaptor,among many envisioned embodiments, and the position of the indexing andidentifier sites (SEQ ID NO: 1). “N” refers to any nucleic acid.

FIG. 4 depicts a schematic of two separate sequence libraries,indicating where the indexing primers and the target primers anneal in atraditional library as compared to a library using an identifier site.

FIG. 5 depicts a data table demonstrating the accuracy of resolving trueduplicates versus apparent or perceived duplicates using an identifiersite in the adaptors.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is based, in part, on compositions and methods fordiscerning duplicate sequencing reads from a population of sequencingreads. The present invention encompasses methods of detecting sequencesduplicated in sequencing applications, and further removal of theduplicated sequence reads. The present invention further encompasseskits comprising components that would allow for customized applicationsof the method of detecting and removing duplicated sequence reads inhigh throughput sequencing reactions. The compositions and methods canbe used with various applications for genetic sample analysis, such asRNA sequence analysis, copy number variation analysis, methylationsequencing analysis, genotyping and whole genome amplification.

Reference will now be made in detail to exemplary embodiments of theinvention. While the disclosed methods and compositions will bedescribed in conjunction with the exemplary embodiments, it will beunderstood that these exemplary embodiments are not intended to limitthe present invention. On the contrary, the present disclosure isintended to encompass alternatives, modifications and equivalents, whichmay be included in the spirit and scope of the present invention.

Unless otherwise specified, terms and symbols of genetics, molecularbiology, biochemistry and nucleic acid used herein follow those ofstandard treatises and texts in the field, e.g. Kornberg and Baker, DNAReplication, Second Edition (W.H. Freeman, New York, 1992); Lehninger,Biochemistry, Second Edition (Worth Publishers, New York, 1975);Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss,New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: APractical Approach (Oxford University Press, New York, 1991); Gait,editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press,Oxford, 1984); and the like.

In some embodiments, the methods disclosed herein is for detecting froma population of sequencing reads a sequencing read, such as a duplicatesequencing read with a duplicate identifier site and target sequence. Aduplicate sequencing read can be a sequencing read with the sameidentifier site and target sequence as another sequencing read in thepopulation of sequencing reads.

Adaptor

The present invention provides compositions of adaptors and methodscomprising use of an adaptor. An adapter refers to an oligonucleotidesequence, the ligation of which to a target polynucleotide or a targetpolynucleotide strand of interest enables the generation ofamplification-ready products of the target polynucleotide or the targetpolynucleotide strand of interest. The target polynucleotide moleculesmay be fragmented or not prior to the addition of adaptors. In someembodiments, a method disclosed herein comprises ligating an adaptor toa 5′ end of each nucleic acid fragment of a plurality of nucleic acidfragments from one or more samples.

Various adaptor designs are envisioned which are suitable for generationof amplification-ready products of target sequence regions/strands ofinterest. For example, the two strands of the adaptor may beself-complementary, non-complementary or partially complementary. Insome embodiments, the adaptor can comprise an indexing primer bindingsite, an indexing site, an identifier site, and a target sequence primerbinding site.

An indexing primer binding site is a nucleotide sequence for binding aprimer for an indexing site. An indexing site is a nucleic acid sequencethat acts as an index for multiple polynucleotide samples, thus allowingfor the samples to be pooled together into a single sequencing run,which is known as multiplexing. In some embodiments, the indexing siteis at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In someembodiments, the indexing site is between 2 and 8 nucleotides in length.In some embodiments, the indexing site is about 6 nucleotides in length.

An identifier site is a nucleic acid sequence that comprises randombases and is used to identify duplicate sequencing reads. In someembodiments, the identifier site is at least 2, 3, 4, 5, 6, 7, 8, 9, or10 nucleotides in length. In some embodiments, the identifier site isbetween 1 and 8 nucleotides in length. In some embodiments, theidentifier site is about 8 nucleotides in length. This identifier sitecan be designed as a set of sequences, or it can be semi-random, or itcan be completely random. In addition, this identifier site can be afixed length, or it can be a variable length. In some embodiments, theidentifier sites in a plurality of adaptors are of a fixed length. Forexample, the identifier sites can all be eight random bases. In anotherembodiment, the identifier sites in a plurality of adaptors are of avariable length. For example, the identifier sites can range in sizefrom 1 to 8 bases. In yet another embodiment, the identifier sites canbe of a defined set of defined sequence. For example, the identifiersite of a plurality of adaptors can be one of 96 defined six-basenucleotide sequences.

A target sequence primer binding site nucleotide sequence for binding aprimer for a target sequence. The primer can be used to amplify thetarget sequence (e.g., a nucleic acid fragment from a sample).Accordingly, in some embodiments, the adaptor comprises an indexingprimer binding site and a target sequence primer binding site.

A primer is a polynucleotide chain, typically less than 200 residueslong, most typically between 15 and 100 nucleotides long, but canencompass longer polynucleotide chains. A primer targeting the primerbinding sites are typically designed to hybridize to single-strandednucleic acid strands. In some embodiments, the primers targeting theprimer binding sites are designed to hybridize to single-stranded DNAtargets. In the case where the sample comprises genomic DNA or otherdouble-stranded DNA, the sample can be first denatured to render thetarget single stranded and enable hybridization of the primers to thedesired sequence regions of interest. In these embodiments, the methodsand compositions described herein can allow for region-specificenrichment and amplification of sequence regions of interest. In someembodiments, the other double-stranded DNA can be double-stranded cDNAgenerated by first and second strand synthesis of one or more targetRNAs.

In other embodiments, the primers targeting the primer binding sites aredesigned to hybridize to double-stranded nucleic acid targets, withoutdenaturation of the double stranded nucleic acids. In other embodiments,the primers targeting the primer binding sites are designed to hybridizeto a double-stranded DNA target, without denaturation of the dsDNA. Inthese embodiments, the primers targeting the selected sequence regionsof interest are designed to form a triple helix (triplex) at theselected sequence regions of interest. The hybridization of the primersto the double-stranded DNA sequence regions of interest can be carriedout without prior denaturation of the double stranded nucleic acidsample. In such embodiments, the methods and compositions describedherein can allow for region-specific enrichment as well asstrand-specific enrichment and amplification of sequence regions ofinterest. This method can be useful for generation of copies of strandspecific sequence regions of interest from complex nucleic acid withoutthe need to denature the dsDNA input DNA, thus enabling enrichment andanalysis of multiplicity of sequence regions of interest in the nativecomplex nucleic acid sample. The method can find use for studies andanalyses carried out in situ, enable studies and analysis of complexgenomic DNA in single cells or collection of very small well definedcell population, as well as permit the analysis of complex genomic DNAwithout disruption of chromatin structures.

The primers of the invention are generally oligonucleotides that areemployed in an extension reaction by a polymerase along a polynucleotidetemplate, such as for amplification of a target sequence (e.g., in PCR).The oligonucleotide primer can be a synthetic polynucleotide that issingle stranded, containing a sequence at its 3′-end that is capable ofhybridizing with a sequence of the target polynucleotide. In someembodiments, the 3′ region of the primer that hybridizes with the targetnucleic acid has at least 80%, preferably 90%, more preferably 95%, mostpreferably 100%, complementarity to a primer binding site.

In some embodiments, the primer binding site is a binding site for auniversal primer. A universal primer is a primer that can be used foramplifying a number of different sequences. In some embodiments, auniversal primer is used to amplify different libraries. In someembodiments, an indexing primer binding site is a binding site for auniversal indexing primer (i.e., the indexing primer binding site is auniversal indexing primer binding site). In some embodiments, theadaptors used for ligating a plurality of nucleic acid fragments have auniversal indexing primer binding site. In some embodiments, theuniversal indexing primer can be used to amplify and/or sequence anumber of different indexing sites.

In some embodiments, a target sequence primer binding site is a bindingsite for a universal target sequence primer (i.e., the target sequenceprimer binding site is a universal target sequence primer binding site).In some embodiments, the adaptors used for ligating a plurality ofnucleic acid fragments have a universal target sequence primer bindingsite. In some embodiments, the universal target sequence primer can beused to amplify and/or sequence a number of different target sequences.

In some embodiments, the adaptor comprises an identifier site 3′ to anindexing site. In some embodiments, the adaptor comprises an identifiersite 5′ to an indexing site. In some embodiments, the adaptor comprisesfrom 5′ to 3′ an indexing primer binding site, indexing site, identifiersite, and target sequence primer binding site. In other embodiments, theadaptor comprises from 5′ to 3′ an indexing primer binding site,indexing site, identifier site, and target sequence primer binding site.In yet other embodiments, the adaptor comprises from 5′ to 3′ anindexing primer binding site, identifier site, indexing site, and targetsequence primer binding site.

Samples

In some embodiments, an adaptor is ligated to a nucleic acid fragment(e.g., the 5′ end of the nucleic acid fragment). The nucleic acidfragment can be from a plurality of nucleic acid fragments from one ormore samples. The nucleic acid fragment can be RNA, DNA, or complex DNA,for example genomic DNA and PNA, in which case one might use a modifiednucleic acid. The nucleic acid fragment may also be cDNA. The cDNA canbe generated from RNA, e.g., mRNA.

The sample can be a biological sample. For example, the sample can be ananimal, plant, bacterial, algal, or viral sample. In some embodiments,the sample is a human, rat, or mouse sample. The sample can be from amixture of genomes of different species such as host-pathogen, bacterialpopulations and the like. The sample can be cDNA made from a mixture ofgenomes of different species. In some embodiments, the sample can befrom a synthetic source. The sample can be mitochondrial DNA. The samplecan be cell-free DNA. The cell-free DNA can be obtained from sourcessuch as a serum or a plasma sample. The sample can comprise one or morechromosomes. For example, if the sample is from a human, the sample cancomprise one or more of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y. In someembodiments, the sample comprises a linear or circular genome. Thesample can be plasmid DNA, cosmid DNA, bacterial artificial chromosome(BAC), or yeast artificial chromosome (YAC). The sample can be from morethan one individual or organism. The sample can be double-stranded orsingle-stranded. The sample can be part of chromatin. The sample can beassociated with histones.

In some embodiments, adaptors are ligated to a plurality of nucleic acidfragments from more than one sample, such as 2, 3, 4, 5, or moresamples. In some embodiments, the nucleic acid fragments from eachsample have the same indexing site. In some embodiments, a plurality ofnucleic acid fragments is generated from a first sample and a secondsample and adaptors are ligated to each nucleic acid fragment, in whichadaptors ligated to each nucleic acid fragment from the first samplehave the same first indexing site and adaptors ligated to nucleic acidfragments from the second sample has the same second indexing site. Insome embodiments, the nucleic acid fragments or data associated with thenucleic acid fragments (e.g., sequencing reads) are separated based onthe indexing site.

In some embodiments, a population of nucleic acid fragments generatedfrom a sample is of one or more specific size range(s). In someembodiments, the fragments have an average length from about 10 to about10,000 nucleotides. In some embodiments, the fragments have an averagelength from about 50 to about 2,000 nucleotides. In some embodiments,the fragments have an average length from about 100-2,500, 10-1,000,10-800, 10-500, 50-500, 50-250, or 50-150 nucleotides. In someembodiments, the fragments have an average length less than 10,000nucleotide, such as less than 5,000 nucleotides, less than 2,500nucleotides, less than 2,500 nucleotides, less than 1,000 nucleotides,less than 500 nucleotides, such as less than 400 nucleotides, less than300 nucleotides, less than 200 nucleotides, or less than 150nucleotides.

In some embodiments, fragmentation of the nucleic acids can be achievedthrough methods known in the art. Fragmentation can be achieved throughphysical fragmentation methods and/or enzymatic fragmentation methods.Physical fragmentation methods can include nebulization, sonication,and/or hydrodynamic shearing. In some embodiments, the fragmentation canbe accomplished mechanically comprising subjecting the nucleic acids inthe input sample to acoustic sonication. In some embodiments, thefragmentation comprises treating the nucleic acids in the input samplewith one or more enzymes under conditions suitable for the one or moreenzymes to generate double-stranded nucleic acid breaks. Examples ofenzymes useful in the generation of nucleic acid or polynucleotidefragments include sequence specific and non-sequence specific nucleases.Non-limiting examples of nucleases include DNase I, Fragmentase,restriction endonucleases, variants thereof, and combinations thereof.Reagents for carrying out enzymatic fragmentation reactions arecommercially available (e.g., from New England Biolabs). For example,digestion with DNase I can induce random double-stranded breaks in DNAin the absence of Mg⁺⁺ and in the presence of Mn⁺⁺. In some embodiments,fragmentation comprises treating the nucleic acids in the input samplewith one or more restriction endonucleases. Fragmentation can producefragments having 5′ overhangs, 3′ overhangs, blunt ends, or acombination thereof. In some embodiments, such as when fragmentationcomprises the use of one or more restriction endonucleases, cleavage ofsample polynucleotides leaves overhangs having a predictable sequence.In some embodiments, the method includes the step of size selecting thefragments via standard methods known in the art such as columnpurification or isolation from an agarose gel.

In some embodiments, fragmentation of the nucleic acids is followed byend repair of the nucleic acid fragments. End repair can include thegeneration of blunt ends, non-blunt ends (i.e sticky or cohesive ends),or single base overhangs such as the addition of a single dA nucleotideto the 3′-end of the nucleic acid fragments, by a polymerase lacking3′-exonuclease activity. End repair can be performed using any number ofenzymes and/or methods known in the art including, but not limited to,commercially available kits such as the Encore™ Ultra Low Input NGSLibrary System I. In some embodiments, end repair can be performed ondouble stranded DNA fragments to produce blunt ends wherein the doublestranded DNA fragments contain 5′ phosphates and 3′ hydroxyls. In someembodiments, the double-stranded DNA fragments can be blunt-end polished(or “end-repaired”) to produce DNA fragments having blunt ends, prior tobeing joined to adapters. Generation of the blunt ends on the doublestranded fragments can be generated by the use of a single strandspecific DNA exonuclease such as for example exonuclease 1, exonuclease7 or a combination thereof to degrade overhanging single stranded endsof the double stranded products. Alternatively, the double stranded DNAfragments can be blunt ended by the use of a single stranded specificDNA endonuclease, for example, but not limited to, mung beanendonuclease or 51 endonuclease. Alternatively, the double strandedproducts can be blunt ended by the use of a polymerase that comprisessingle stranded exonuclease activity such as for example T4 DNApolymerase, or any other polymerase comprising single strandedexonuclease activity or a combination thereof to degrade the overhangingsingle stranded ends of the double stranded products. In some cases, thepolymerase comprising single stranded exonuclease activity can beincubated in a reaction mixture that does or does not comprise one ormore dNTPs. In other cases, a combination of single stranded nucleicacid specific exonucleases and one or more polymerases can be used toblunt end the double stranded fragments generated by fragmenting thesample comprising nucleic acids. In still other cases, the nucleic acidfragments can be made blunt ended by filling in the overhanging singlestranded ends of the double stranded fragments. For example, thefragments may be incubated with a polymerase such as T4 DNA polymeraseor Klenow polymerase or a combination thereof in the presence of one ormore dNTPs to fill in the single stranded portions of the doublestranded fragments. Alternatively, the double stranded DNA fragments canbe made blunt by a combination of a single stranded overhang degradationreaction using exonucleases and/or polymerases, and a fill-in reactionusing one or more polymerases in the presence of one or more dNTPs.

U.S. Patent Publication Nos. 2013-0231253 A1 and 2014-0274729 A1 furtherdescribe methods of generating nucleic acid fragments, methods ofmodifying the fragments and analysis of the fragments, and areincorporated by reference in their entirety herein.

Ligation of Adaptors

Ligation of adaptors at the desired end of the sequence regions ofinterest (e.g., at the 5′ or 3′ end of a nucleic acid fragment generatedform a sample) is suitable for carrying out the methods of theinvention. Various ligation modalities are envisioned, dependent on thechoice of nucleic acid, nucleic acid modifying enzymes and the resultingligatable end of the nucleic acid. For example, when a blunt end productcomprising the target region/sequence of interest is generated, bluntend ligation can be suitable. Alternatively, where the cleavage iscarried out using a restriction enzyme of known sequence specificity,leading to the generation of cleavage sites with known sequenceoverhangs, suitable ends of the adaptors can be designed to enablehybridization of the adaptor to the cleavage site of the sequence regionof interest and subsequent ligation. Ligation also refers to any joiningof two nucleic acid molecules that results in a single nucleic acidsequence that can be further modified to obtain the sequence of thenucleic acids in question. Reagents and methods for efficient and rapidligation of adaptors are commercially available, and are known in theart.

In some embodiments, the 5′ and/or 3′ end nucleotide sequences offragmented nucleic acids are not modified or end-repaired prior toligation with the adapter oligonucleotides of the present invention. Forexample, fragmentation by a restriction endonuclease can be used toleave a predictable overhang, followed by ligation with one or moreadapter oligonucleotides comprising an overhang complementary to thepredictable overhang on a nucleic acid fragment. In another example,cleavage by an enzyme that leaves a predictable blunt end can befollowed by ligation of blunt-ended nucleic acid fragments to adapteroligonucleotides comprising a blunt end. In some embodiments, end repaircan be followed by an addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides, such as one or moreadenine, one or more thymine, one or more guanine, or one or morecytosine, to produce an overhang. Nucleic acid fragments having anoverhang can be joined to one or more adapter oligonucleotides having acomplementary overhang, such as in a ligation reaction. For example, asingle adenine can be added to the 3′ ends of end-repaired DNA fragmentsusing a template independent polymerase, followed by ligation to one ormore adapters each having a thymine at a 3′ end. In some embodiments,adapter oligonucleotides can be joined to blunt end double-strandednucleic acid fragments which have been modified by extension of the 3′end with one or more nucleotides followed by 5′ phosphorylation. In somecases, extension of the 3′ end can be performed with a polymerase suchas for example Klenow polymerase or any of the suitable polymerasesprovided herein, or by use of a terminal deoxynucleotide transferase, inthe presence of one or more dNTPs in a suitable buffer containingmagnesium. In some embodiments, nucleic acid fragments having blunt endscan be joined to one or more adapters comprising a blunt end.Phosphorylation of 5′ ends of nucleic acid fragments can be performedfor example with T4 polynucleotide kinase in a suitable buffercontaining ATP and magnesium. The fragmented nucleic acid molecules mayoptionally be treated to dephosphorylate 5′ ends or 3′ ends, forexample, by using enzymes known in the art, such as phosphatases.

In some embodiments, appending the adaptor to the nucleic acid fragmentsgenerated by methods described herein can be achieved using a ligationreaction or a priming reaction. In some embodiments, appendage of anadaptor to the nucleic acid fragments comprises ligation. In someembodiments, ligation of the adaptor to the nucleic acid fragments canbe following end repair of the nucleic acid fragments. In anotherembodiment, the ligation of the adaptor to the nucleic acid fragmentscan be following generation of the nucleic acid fragments without endrepair of the nucleic acid fragments. The adaptor can be any type ofadaptor known in the art including, but not limited to, conventionalduplex or double stranded adaptors in which the adaptor comprises twocomplementary strands. In some embodiments, the adaptor can be a doublestranded DNA adaptor. In some embodiments, the adaptor can be anoligonucleotide of known sequence and, thus, allow generation and/or useof sequence specific primers for amplification and/or sequencing of anypolynucleotides to which the adaptor is appended or attached. In someembodiments, the adaptor can be a conventional duplex adaptor, whereinthe adaptor comprises sequence well known in the art. In a someembodiments, the adaptor can be appended to the nucleic acid fragmentsgenerated by the methods described herein in multiple orientations. Insome embodiment, the methods described herein can involve the use of aduplex adaptor comprising double stranded DNA of known sequence that isblunt ended and can bind to the double stranded nucleic acid fragmentsgenerated by the methods described herein in one of two orientations. Insome embodiments, the adaptor can be ligated to each of the nucleic acidfragments such that each of the nucleic acid fragments comprises thesame adaptor. In other words, each of the nucleic acid fragmentscomprises a common adaptor. In another embodiment, an adaptor can beappended or ligated to a library of nucleic acid fragments generated bythe methods described herein such that each nucleic acid fragment in thelibrary of nucleic acid fragments comprises the adaptor ligated to oneor both ends. In another embodiment, more than one adaptor can beappended or ligated to a library of nucleic acid fragments generated bythe methods described herein. The multiple adaptors may occur adjacentto one another, spaced intermittently, or at opposite ends of thenucleic acid fragments. In some embodiments, the adaptor can be ligatedor appended to the 5′ and/or 3′ ends of the nucleic acid fragmentsgenerated by the methods described herein. The adaptor can comprise twostrands wherein each strand comprises a free 3′ hydroxyl group butneither strand comprises a free 5′ phosphate. In some embodiments, thefree 3′ hydroxyl group on each strand of the adaptor can be ligated to afree 5′ phosphate present on either end of the nucleic acid fragments ofthe present invention. In this embodiment, the adaptor comprises aligation strand and a non-ligation strand whereby the ligation strandcan be ligated to the 5′ phosphate on either end of the nucleic acidfragment while a nick or gap can be present between the non-ligationstrand of the adaptor and the 3′ hydroxyl on either end of the nucleicacid fragment. In some embodiments, the nick or gap can be filled in byperforming a gap repair reaction. In some embodiments, the gap repaircan be performed with a DNA dependent DNA polymerase with stranddisplacement activity. In some embodiments, the gap repair can beperformed using a DNA-dependent DNA polymerase with weak or no stranddisplacement activity. In some embodiments, the ligation strand of theadaptor can serve as the template for the gap repair or fill-inreaction. The gap repair or fill-in reaction may comprise an extensionreaction wherein the ligation strand of the adaptor serves as a templateand leads to the generation of nucleic acid fragments with complementarytermini or ends. In some embodiments, the gap repair can be performedusing Taq DNA polymerase. In some embodiments, the ligation of the firstadaptor to the nucleic acid fragments generated by the methods describedherein may not be followed by gap repair. The nucleic acid fragments maycomprise the adaptor sequence ligated only at the 5′ end of each strand.

Ligation and, optionally gap repair, of the adaptor to the nucleic acidfragments generates an adaptor-nucleic acid fragment complex. In someembodiments, the adaptor-nucleic acid fragment complex can be denatured.Denaturation can be achieved using any of the methods known in the artincluding, but not limited to, physical, thermal, and/or chemicaldenaturation. In some embodiments, denaturation can be achieved usingthermal or heat denaturation. In some embodiments, denaturation of theadaptor-nucleic acid fragment complex generates single stranded nucleicacid fragments comprising the adaptor sequence at only the 5′end of thenucleic acid fragments. In another embodiment, denaturation of the firstadaptor-nucleic acid fragment complex generates single stranded nucleicacid fragments comprising adaptor sequence at both the 5′end and 3′endof the nucleic acid fragments.

Methods of Amplification

The methods, compositions and kits described herein can be useful togenerate amplification-ready products directly from a nucleic acidsource for downstream applications such as next generation sequencing,as well as generation of libraries with enriched population of sequenceregions of interest. In some embodiments, the adapter-nucleic fragmentligated products, e.g., from the ligation of an adaptor to a 5′ end ofeach nucleic acid fragment of a plurality of nucleic acid fragments fromone or more samples, is amplified.

Methods of amplification are well known in the art. In some embodiments,the amplification is exponential, e.g. in the enzymatic amplification ofspecific double stranded sequences of DNA by a polymerase chain reaction(PCR). In other embodiments the amplification method is linear. In otherembodiments the amplification method is isothermal. In some embodiments,the amplification is exponential, e.g. in the enzymatic amplification ofspecific double stranded sequences of DNA by a polymerase chain reaction(PCR).

Suitable amplification reactions can be exponential or isothermal andcan include any DNA amplification reaction, including but not limited topolymerase chain reaction (PCR), strand displacement amplification (SDA)linear amplification, multiple displacement amplification (MDA), rollingcircle amplification (RCA), single primer isothermal amplification(SPIA, see e.g. U.S. Pat. No. 6,251,639), Ribo-SPIA, or a combinationthereof. In some cases, the amplification methods for providing thetemplate nucleic acid may be performed under limiting conditions suchthat only a few rounds of amplification (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2.4, 25, 26,28, 29, 30 etc.), such as for example as is commonly done for cDNAgeneration, The number of rounds of amplification can be about 1-30,1-20, 1-1-10, 5-30, 10-30, 15-30, 20-30, 10-30, 15-30, 20-30, or 25-30.

PCR is an in vitro amplification procedure based on repeated cycles ofdenaturation, oligonucleotide primer annealing, and primer extension bythermophilic template dependent polynucleotide polymerase, resulting inthe exponential increase in copies of the desired sequence of thepolynucleotide analyte flanked by the primers. The two different PCRprimers, which anneal to opposite strands of the DNA, are positioned sothat the polymerase catalyzed extension product of one primer can serveas a template strand for the other, leading to the accumulation of adiscrete double stranded fragment whose length is defined by thedistance between the 5′ ends of the oligonucleotide primers.

LCR uses a ligase enzyme to join pairs of preformed nucleic acid probes.The probes hybridize with each complementary strand of the nucleic acidanalyte, if present, and ligase is employed to bind each pair of probestogether resulting in two templates that can serve in the next cycle toreiterate the particular nucleic acid sequence.

SDA (Westin et a 2000, Nature Biotechnology, 18, 199-202; Walker et al1992, Nucleic Acids Research, 20, 7, 1691-1696), is an isothermalamplification technique based upon the ability of a restrictionendonuclease such as HincII or BsoBI to nick the unmodified strand of ahemiphosphorothioate form of its recognition site, and the ability of anexonuclease deficient DNA polymerase such as Klenow exo minuspolymerase, or Bst polymerase, to extend the 3′-end at the nick anddisplace the downstream DNA strand. Exponential amplification resultsfrom coupling sense and antisense reactions in which strands displacedfrom a sense reaction serve as targets for an antisense reaction andvice versa.

Some aspects of the invention utilize linear amplification of nucleicacids or polynucleotides. Linear amplification generally refers to amethod that involves the formation of one or more copies of thecomplement of only one strand of a nucleic acid or polynucleotidemolecule, usually a nucleic acid or polynucleotide analyte. Thus, theprimary difference between linear amplification and exponentialamplification is that in the latter process, the product serves assubstrate for the formation of more product, whereas in the formerprocess the starting sequence is the substrate for the formation ofproduct but the product of the reaction, i.e. the replication of thestarting template, is not a substrate for generation of products. Inlinear amplification the amount of product formed increases as a linearfunction of time as opposed to exponential amplification where theamount of product formed is an exponential function of time.

Downstream Applications

One aspect of the present invention is that the methods and compositionsdisclosed herein can be efficiently and cost-effectively utilized fordownstream analyses, such as in next generation sequencing orhybridization platforms, with minimal loss of biological material ofinterest. The methods of the present invention can also be used in theanalysis of genetic information of selective genomic regions of interest(e.g., analysis of SNPs or other disease markers) as well as genomicregions which may interact with the selective region of interest. Themethods of the present invention may further be used in the analysis ofcopy number variation as well as differential expression.

Sequencing

In some embodiments, a population of sequencing reads is generated fromthe amplified adapter-nucleic fragment ligated products. In someembodiments, a sequencing read comprises an index read, which comprisesthe sequence of an indexing site. In some embodiments, an index readcomprises the sequence of an indexing site and the sequence of anidentifier site. For example, the indexing site is sequenced with theidentifier site. In some embodiments, the index read does not includethe sequence of an identifier site. For example, the indexing site isnot sequenced with the identifier site. In some embodiments, asequencing read comprises the target sequence. In some embodiments, asequencing read comprises a target sequence and an identifier sequence.For example, the target sequence is sequenced with the identifier site.In some embodiments, the target sequence is not sequenced with theidentifier site.

The methods of the present invention are useful for sequencing by themethod commercialized by Illumina, as described U.S. Pat. Nos.5,750,341; 6,306,597; and 5,969,119.

In general, double stranded fragment polynucleotides can be prepared bythe methods of the present invention to produce amplified nucleic acidsequences tagged at one (e.g., (A)/(A′) or both ends (e.g., (A)/(A′) and(C)/(C′)). In some cases, single stranded nucleic acid tagged at one orboth ends is amplified by the methods of the present invention (e.g., bySPIA or linear PCR). The resulting nucleic acid is then denatured andthe single-stranded amplified polynucleotides are randomly attached tothe inside surface of flow-cell channels. Unlabeled nucleotides areadded to initiate solid-phase bridge amplification to produce denseclusters of double-stranded DNA. To initiate the first base sequencingcycle, four labeled reversible terminators, primers, and DNA polymeraseare added. After laser excitation, fluorescence from each cluster on theflow cell is imaged. The identity of the first base for each cluster isthen recorded. Cycles of sequencing are performed to determine thefragment sequence one base at a time.

In some embodiments, the methods of the invention are useful forpreparing target polynucleotides for sequencing by the sequencing byligation methods commercialized by Applied Biosystems (e.g., SOLiDsequencing). In other embodiments, the methods are useful for preparingtarget polynucleotides for sequencing by synthesis using the methodscommercialized by 454/Roche Life Sciences, including but not limited tothe methods and apparatus described in Margulies et al., Nature (2005)437:376-380 (2005); and U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390;7,244,567; 7,264,929; and 7,323,305. In other embodiments, the methodsare useful for preparing target polynucleotide(s) for sequencing by themethods commercialized by Helicos BioSciences Corporation (Cambridge,Mass.) as described in U.S. application Ser. No. 11/167,046, and U.S.Pat. Nos. 7,501,245; 7,491,498; 7,276,720; and in U.S. PatentApplication Publication Nos. US20090061439; US20080087826;US20060286566; US20060024711; US20060024678; US20080213770; andUS20080103058. In other embodiments, the methods are useful forpreparing target polynucleotide(s) for sequencing by the methodscommercialized by Pacific Biosciences as described in U.S. Pat. Nos.7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503;7,315,019; 7,302,146; 7,313,308; and US Application Publication Nos.US20090029385; US20090068655; US20090024331; and US20080206764.

An example of a sequencing technique that can be used in the methods ofthe provided invention is semiconductor sequencing provided by IonTorrent (e.g., using the Ion Personal Genome Machine (PGM)). Ion Torrenttechnology can use a semiconductor chip with multiple layers, e.g., alayer with micro-machined wells, an ion-sensitive layer, and an ionsensor layer. Nucleic acids can be introduced into the wells, e.g., aclonal population of single nucleic can be attached to a single head,and the bead can be introduced into a well. To initiate sequencing ofthe nucleic acids on the beads, one type of deoxyribonucleotide (e.g.,dATP, dCTP, dGTP, or dTTP) can be introduced into the wells. When one ormore nucleotides are incorporated by DNA polymerase, protons (hydrogenions) are released in the well, which can be detected by the ion sensor.The semiconductor chip can then be washed and the process can berepeated with a different deoxyribonucleotide. A plurality of nucleicacids can be sequenced in the wells of a semiconductor chip. Thesemiconductor chip can comprise chemical-sensitive field effecttransistor (chemFET) arrays to sequence DNA (for example, as describedin U.S. Patent Application Publication No. 20090026082). Incorporationof one or more triphosphates into a new nucleic acid strand at the 3′end of the sequencing primer can be detected by a change in current by achemFET. An array can have multiple chemFET sensors.

Another example of a sequencing technique that can be used in themethods of the provided invention is nanopore sequencing (see e.g., SoniG V and Meller A. (2007) Clin Chem 53: 1996-2001). A nanopore can be asmall hole of the order of 1 nanometer in diameter. Immersion of ananopore in a conducting fluid and application of a potential across itcan result in a slight electrical current due to conduction of ionsthrough the nanopore. The amount of current that flows is sensitive tothe size of the nanopore. As a DNA molecule passes through a nanopore,each nucleotide on the DNA molecule obstructs the nanopore to adifferent degree. Thus, the change in the current passing through thenanopore as the DNA molecule passes through the nanopore can represent areading of the DNA sequence.

Data Analysis

In some embodiments, the sequence reads are used in the analysis ofgenetic information of selective genomic regions of interest as well asgenomic regions which may interact with the selective region ofinterest. Amplification methods as disclosed herein can be used in thedevices, kits, and methods known to the art for genetic analysis, suchas, but not limited to those found in U.S. Pat. Nos. 6,449,562,6,287,766, 7,361,468, 7,414,117, 6,225,109, and 6,110,709.

In some embodiments, the sequencing reads are used to detect duplicatesequencing reads. In some embodiments, a sequencing read is identifiedas a duplicate sequencing read when it contains an identifier site andtarget sequence that is the same as another sequencing read from thesame population of sequencing reads.

In some embodiments, duplicate sequencing reads are differentiated fromone another as being true duplicates versus apparent or perceivedduplicates. Apparent or perceived duplicates may be identified fromsequencing libraries and using conventional measures of duplicate reads(i.e., reads were mapped using bowtie), where all reads with the samestart and end nucleic acid coordinates were counted as duplicates. Trueduplicates may be identified from sequencing libraries having had anidentifier site introduced through ligation to differentiate betweenfragments of DNA that randomly have the same start and end mappingcoordinates.

In some embodiments, the sequence of the identifier sites from the indexread of two nucleic acids generated from any dsDNA may have the samestart site, as determined from the sequencing reads of the targetsequence of the generated nucleic acid fragments. If the identifier sitefrom the index read of the two nucleic acid fragments are not identical,then the target sequence reads were not generated from the same originaldsDNA molecule and, therefore, are no true duplicate reads. The ligationof random sequences onto dsDNA molecules, accompanied by the methods ofthe invention, allow for the identification of true duplicate readsversus apparent or perceived duplicate reads.

In some embodiments, the sequence of the identifier sites from the indexread of two nucleic acid fragments generated from a genomic DNA (gDNA)molecule that have the same start site, which can be determined from thesequencing reads of the target sequence of the nucleic acid fragments isdetermined. If the identifier site from the index read of the twonucleic acid fragments are not identical, then the target sequence readswere not generated from the same original gDNA molecule and, therefore,are not true duplicate reads. In another embodiment, an identifier siteis inserted at the adapter insert junction. The sequence of theidentifier site is carried through the library amplification step. Theidentifier site is the first sequence read during the forward read. Asthe identifier sequence is not logically present adjacent to naturallyoccurring sequence, this uniquely identifies the DNA fragment.Therefore, by ligating random sequences onto the original gDNA, themethods of the invention identify true duplicate reads.

In some embodiments, a duplicate sequencing read is detected andanalyzed. Duplicate reads can be filtered using ‘samtools rmdup’,wherein reads with identical external coordinates are removed, only oneread with highest mapping quality is retained. After filtering, afiltered set of deduplicated reads can be used in any downstreamanalysis. Conversely, this filtering step can be skipped, and downstreamanalysis can be done using the unfiltered reads, including duplicates.

In some embodiments, sequencing reads are generated from a number ofsamples. In some embodiments, adaptors are ligated to a plurality ofnucleic acid fragments from the samples, in which the nucleic acidfragments from each sample has the same indexing site. In someembodiments, a plurality of nucleic acid fragments is generated from afirst sample and a second sample and adaptors are ligated to eachnucleic acid fragment, in which adaptors ligated to each nucleic acidfragment from the first sample have the same first indexing site andadaptors ligated to nucleic acid fragments from the second sample hasthe same second indexing site. In some embodiments, the data associatedwith the nucleic acid fragments (e.g., sequencing reads) are separatedbased on the indexing site before sequencing reads of the targetsequence and/or identifier site are analyzed. In some embodiments, thenucleic acid fragments or data associated with the nucleic acidfragments (e.g., sequencing reads) are separated based on the indexingsite before duplicate sequencing reads are analyzed and/or removed.

In some embodiments, a method disclosed herein identifies or detects oneor more true duplicate(s) with increased accuracy as compared to othermethods. For example, in some embodiments, a method disclosed hereinidentify true duplicates (in contrast to identifying apparent orperceived duplicates) with increased accuracy as compared to othermethods. The increased resolution and/or accuracy in identifying one ormore true duplicate(s) can provide a considerable contribution to thestate of the art in more accurately identifying true duplicates. In someembodiments, a method disclosed herein identifies or detects a trueduplicate with increased efficiency as compared to other methods, suchas paired end sequencing. The increase in accuracy, resolution, and/orefficiency in detecting duplicate reads (e.g., true duplicate(s)) canincrease confidence in sequencing results, such as for expression andCNV analysis.

Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, the kit, in a suitable container, comprises: anadaptor or several adaptors, one or more of oligonucleotide primers andreagents for amplification.

The containers of the kits will generally include at least one vial,test tube, flask, bottle, syringe or other containers, into which acomponent may be placed, and preferably, suitably aliquotted. Wherethere is more than one component in the kit, the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a container.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution. However, thecomponents of the kit may be provided as dried powder(s). When reagentsand/or components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent.

A kit may include instructions for employing the kit components as wellthe use of any other reagent not included in the kit. Instructions mayinclude variations that can be implemented.

In some embodiments, the invention provides kits containing any one ormore of the elements disclosed in the above methods and compositions. Insome embodiments, a kit comprises a composition of the invention, in oneor more containers. In some embodiments, the invention provides kitscomprising adapters, primers, and/or other oligonucleotides describedherein. In some embodiments, the kit further comprises one or more of:(a) a DNA ligase, (b) a DNA-dependent DNA polymerase, (c) anRNA-dependent DNA polymerase, (d) a forward adapter (e) one or moreoligonucleotides comprising reverse adaptor sequence and (f) one or morebuffers suitable for one or more of the elements contained in said kit.The adapters, primers, other oligonucleotides, and reagents can be,without limitation, any of those described above. Elements of the kitcan further be provided, without limitation, in any of the amountsand/or combinations (such as in the same kit or same container)described above. The kits may further comprise additional agents, suchas those described above, for use according to the methods of theinvention. For example, the kit can comprise a first forward adaptorthat is a partial duplex adaptor as described herein, a second forwardadapter, and a nucleic acid modifying enzyme specific for a restrictionand/or cleavage site present in the first forward adaptor. The kitelements can be provided in any suitable container, including but notlimited to test tubes, vials, flasks, bottles, ampules, syringes, or thelike. The agents can be provided in a form that may be directly used inthe methods of the invention, or in a form that requires preparationprior to use, such as in the reconstitution of lyophilized agents.Agents may be provided in aliquots for single-use or as stocks fromwhich multiple uses, such as in a number of reaction, may be obtained.

In some embodiments, the kit comprises a plurality of adaptoroligonucleotides, wherein each of the adaptor oligonucleotides comprisesat least one of a plurality of identifier site sequences, wherein eachidentifier site sequence of the plurality of identifier site sequencesdiffers from every other identifier site sequence in said plurality ofidentifier site sequences at at least three nucleotide positions, andinstructions for using the same. Adapters comprising differentidentifier site sequences can be supplied individually or in combinationwith one or more additional adapters having a different identifier sitesequence. In some embodiments, the kit can comprises a plurality ofadapter oligonucleotides.

Examples Example 1: Identification of Duplicate Sequencing Reads withNuGEN Ovation Target Enrichment Library System

Sample Description:

100 ng of DNA from a human HapMap sample (NA19238) was fragmented toapproximately 500 base pair in length by sonication with a Covarissystem (Covaris, Inc., Woburn, Mass.). The resulting DNA was treatedwith end repair enzyme mix NuGEN R01280 and R01439 (NuGEN Technologies,Inc., San Carlos, Calif.) according to supplier's recommendation toproduce blunt ended DNA fragments.

Library Generation, Enrichment, and Identifier Site Incorporation:

An oligonucleotide with segments, from 5′ to 3′ of the top strand, 1) anIllumina indexing read priming site such as AGAGCACACGTCTGAACTCCAGTCAC(SEQ ID NO:2), 2) an indexing site, 3) an identifier site having arandom 6 base sequence and, 4) a sequence compatible with an Illuminaforward sequencing priming site such as TCTTTCCCTACACGACGCTCTTCCGATCT(SEQ ID NO:3), was annealed to a second oligonucleotide to form apartially double stranded DNA adapter. Five uM of these adapters wereligated onto the end-repaired DNA using Ligase and Ligase reactionbuffer from NuGEN's Ovation Ultralow Library System (NuGEN Technologies,Inc., San Carlos, Calif.) according to supplier's recommendations.Following 30 minutes of incubation at 25° C., the reaction mixture wasdiluted with water, 0.8× volume of Ampure XP magnetic beads (AgencourtBiosciences Corporation, A Beckman Coulter Company, Beverly, Mass.) wasadded and the solution thoroughly mixed. The beads were collected,washed and the ligated DNA fragments eluted according to manufacturer'srecommendations. A pool of targeting probes was annealed to the elutedDNA fragments by initially heating the solution to 95° C. then slowlycooling the mixture from 80° C. to 60° C. by 0.6 degrees/minute.Targeting probes that were specifically annealed were extended with TaqDNA polymerase (New England Biolabs, Inc., Ipswich, Mass.) according tothe manufacturer's protocols. Following extension, the DNA fragmentswere collected on Agencourt magnetic beads, washed and eluted accordingto manufacturer's recommendations. These libraries were enriched by 30cycles of PCR using NuGEN library enrichment primers (Ovation TargetEnrichment Library System, NuGEN Technologies, Inc., San Carlos, Calif.)that also contain the Illumina flow cell sequences (Illumina Inc., SanDiego, Calif.) according to supplier's recommendation.

The resulting libraries were quantitated by qPCR using a kit provided byKAPA, diluted to 2 nM and applied to an Illumina MiSeq DNA Sequencer(Illumina Inc., San Diego, Calif.). The following series was run: 36base first read, 14 base second read, and 24 base third read.

Data Analysis: The sequencer output was processed in accordance withmanufacturer's recommendation. In order to analyze the data, theindexing read was split into two files. The first file contained thefirst 8 bases of the indexing reads and is utilized as the library indexfile for standard library parsing. The other file contains only therandom bases and is set aside for further sequence parsing.

Following our data analysis pipeline of sequence alignment with bowtiealigner (Langmead B. et al., Ultrafast and memory-efficient alignment ofshort DNA sequences to the human genome. Genome Biol. 2009, 10:R2.),duplicate reads were identified by their genomic start positions. Atthis point, sequencing reads that started at the same genomic positionwere checked against the random bases file to see if they have the sameor different set of random bases that were ligated to them. Where twosequences with the same starting genomic coordinate had the same set ofrandom bases, they were considered to have come from the same initialDNA ligation event, regardless of which Ovation Target Enrichmenttargeting probe was used to generate the sequence fragment in question.These sequences, therefore, did not provide unique information about thestarting genomic DNA and are considered as one sequencing read for thepurpose of variant analysis. Two sequence reads with the same startinggenomic coordinate that have different random bases were derived fromunique ligation events and were considered to both be valid sequencingreads for the purpose of variant identification. FIG. 5 provides theanalysis results demonstrating the identification of the duplicatereads. The use of an identifier site allowed for the determination ofthe number of true duplications versus the number of apparent orperceived duplicates.

If the sequences of two libraries are identical, their duplicationstatus is unknown since this could occur by chance in any library. If anidentifier sequence is used in combination with the library reads, thestatus can be determined (duplicates if identical, distinct ifdifferent). With the SPET system, one end is common so the probabilityof two libraries having identical ends increases. These would appear tobe duplicate sequences and their true status can be determined bylooking at an identifier sequence.

Over the sampling of all randomly selected reads, the use of identifiersites provided an increased resolution on the presence of trueduplicates. When evaluating two million random reads, the apparentduplicates were found to comprise 39% of all reads. However, the trueduplicates, identified through the use of an identifier site, were foundto comprise only 26% of all reads. The methods employing the use of anidentifier site were found to considerably increase the resolution ofthe true number of duplicates within the pool of reads.

Example 2: Removal of Duplicate Sequencing Reads with 8 Base IdentifierSite

In a standard RNA sequencing library, adaptors are ligated to the endsof double stranded cDNA. These adaptors contain universal sequences thatallow for PCR amplification and sequencing on high throughput sequencingmachines. The adaptors are synthesized with a large population ofadditional sequences, in which each additional sequence is an identifiersite, at the ligation end. The identifier sites are present at thejunction between the adaptor and the cDNA. The sequence read starts withthe identifier site and follows with the cDNA sequence.

This pool of identifier sites is used for the detection of PCRduplicates, as PCR duplicates will contain the identical identifiersite, whereas two different cDNA molecules will ligate to two differentadaptors containing two different identifier sites. This identifier siteis designed as eight random bases introduced onto the end of theadaptor. Sequence reads from libraries made with such adaptors containthe 8 bases of the identifier site followed by the cDNA sequence.Standard PCR duplicate removal software such as PICARD, Markduplicates,and/or SAMtools rndup is used to identify and remove the PCR duplicates,leaving behind for analysis any instances of multiple cDNA fragmentsthat happen to have the same sequence.

Example 3: Removal of Duplicate Sequencing Reads with Mixture of Random1-8 Base Identifier Sites

In a standard RNA sequencing library, adaptors are ligated to the endsof double stranded cDNA. These adaptors contain universal sequences thatallow for PCR amplification and sequencing on high throughput sequencingmachines. The adaptors are synthesized with a large population ofadditional sequences, in which each additional sequence is an identifiersite, at the ligation end. The identifier sites are present at thejunction between the adaptor and the cDNA. The sequence read starts withthe identifier site and follows with the cDNA sequence.

This pool of identifier sites is used for the detection of PCRduplicates, as PCR duplicates will contain the identical identifiersite, whereas two different cDNA molecules will ligate to two differentadaptors containing two different identifier sites.

One to eight random bases are introduced onto the end of the adaptor.Sequence reads from libraries made with such adaptors contain between 1and 8 bases of the identifier site followed by the cDNA sequence.Standard PCR duplicate removal software such as PICARD, Markduplicates,and/or SAMtools rndup is used to identify and remove the PCR duplicates,leaving behind for analysis any instances of multiple cDNA fragmentsthat happen to have the same sequence.

Example 4: Removal of Duplicate Sequencing Reads with Mixture of 96Defined 6-Base Identifier Sites

In a standard RNA sequencing library, adaptors are ligated to the endsof double stranded cDNA. These adaptors contain universal sequences thatallow for PCR amplification and sequencing on high throughput sequencingmachines. The adaptors are synthesized with a large population ofadditional sequences, in which each additional sequence is an identifiersite, at the ligation end. The identifier sites are present at thejunction between the adaptor and the cDNA. The sequence read starts withthe identifier site and follows with the cDNA sequence.

This pool of identifier sites is used for the detection of PCRduplicates, as PCR duplicates will contain the identical identifiersite, whereas two different cDNA molecules will ligate to two differentadaptors containing two different identifier sites.

A mixture of 96 defined six-bases sequences are introduced onto the endof the adaptors. Thus, each six-base sequence is an identifier site.Sequence reads from libraries made with such adaptors contain one of the96 six-base identifier site followed by the cDNA sequence. Standard PCRduplicate removal software such as PICARD, Markduplicates, and/orSAMtools rndup is used to identify and remove the PCR duplicates,leaving behind for analysis any instances of multiple cDNA fragmentsthat happen to have the same sequence.

Example 5: Identification of Duplicate Sequencing Reads in DeterminingmRNA Expression Levels

Sample Description: Total RNA is extracted from tumor and normaladjacent tissue for the purpose of finding differences in expressionlevels of transcripts between the two sample types. 100 ng of eachsample is converted into cDNA using the USP primers, reaction buffer,and Reverse Transcriptase provided in NuGEN's Encore Complete LibrarySystem (NuGEN Technologies, Inc., San Carlos, Calif.) according to thesupplier's recommendations. This is followed by second strand synthesis,again using materials provided in the kit according to recommendations.Double stranded cDNA was prepared using the SuperScript® Double-StrandedcDNA Synthesis Kit from Life Technologies (Carlsbad, Calif.) accordingthe manufacturer's instructions. DNA was sheared with a Covaris S-seriesdevice (Covaris, Inc., Woburn, Mass.) using the 200 bp sonicationprotocol provided with the instrument (10% duty cycle, 200 cycles/burst,5 intensity, 180 seconds). DNA was treated with 1.5 μL 10× BluntingBuffer, 0.5 uL Blunting Enzyme (New England Biolabs, Inc., Ipswich,Mass.; p/n E1201) and 1.2 uL of 2.5 mM of each dNTP mix in a totalvolume of 15 uL for 30 minutes at 25° C. followed by 10 minutes at 70°C.

Library Generation:

The DNA fragments were then subjected to end repair using end repairbuffers and enzymes provided in NuGEN's Ovation Ultralow Library System(NuGEN Technologies, Inc., San Carlos, Calif.).

Forward adaptor (SEQ ID NO: 4) 5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCGATCTNNNNNNNN,Reverse adaptor 1) (SEQ ID NO: 5) 5′CAAGCAGAAGACGGCATACGAGATTCCCTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN, Reverse adaptor 2) (SEQ ID NO: 6) 5′CAAGCAGAAGACGGCATACGAGATTGAAGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNNNNNNNN, and Common partner (SEQ ID NO: 7) 5′NNNNNNNNAGATCGGAAGAGCand

Common partner 5′ NNNNNNNNAGATCGGAAGAGC (SEQ ID NO:7) were all orderedfrom IDT (Integrated DNA Technologies, Coralville, Iowa). The reverseadaptors each contain a unique identifier (underlined) that enableslibraries made with these adapters to be distinguished. In this case Nrepresents an equimolar mixture of A, C, G, and T. A mixture of 5 uMforward, 5 uM reverse, and 10 uM common in 10 mM MgCl2, 50 mM Tris pH 8was heated to 95 C for 5 minutes, then cooled to 20 C. Adaptor ligationwas performed by addition of 4.5 uL water, 3 uL Adaptor mix (preparedabove), 6 uL 5× NEBNext Quick Ligation Reaction Buffer and 1.5 uL QuickT4 DNA Ligase (New England Biolabs, Inc., Ipswich, Mass.; p/n E6056),followed by incubation for 30 minutes at 25° C. followed by 10 minutesat 70° C. Ligation products were purified by adding 70 uL water and 80uL of Ampure XP beads (Agencourt Genomics), washing twice with 70%ethanol and eluting with 20 uL of 10 mM Tris pH 8.0. Library productswere amplified in a 50 uL PCR containing 0.5 uM each of primer (5′AATGATACGGCGACCACCGA (SEQ ID NO:8), and 5′ CAAGCAGAAGACGGCATACGA (SEQ IDNO:9), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.2 mM each dNTP,and 1 unit Taq polymerase. The reaction was cycled 15 times under theconditions 95 C for 15 seconds, 60 C for 1 minute. PCR products werepurified with 1 volume of Ampure XP beads (Agencourt BiosciencesCorporation, A Beckman Coulter Company, Beverly, Mass.) as describedabove. The library was analyzed by HS DNA Bioanalyzer (AgilentTechnologies, Santa Clara, Calif.) and quantitated with the KAPA LibraryQuantification Kit (KAPA Biosystems, Wilmington, Mass.; p/n KK4835)according to the supplied instructions. The resulting libraries arecombined and are compatible with standard TruSeq single end or pairedIllumina sequencing protocols for GAIIx, MiSeq, or Hi Seq sequencinginstruments (Illumina Inc., San Diego, Calif.). The following series isrun; 50 base first read, 6 base second read. A third read is notrequired for counting purposes or duplication analysis.

Data Analysis:

The sequencer output was processed in accordance with manufacturer'srecommendation. The 6 bases of the index read is used for standardlibrary parsing, separating the data files from the two sample types.The first 50 bases of the target sequence read are compared to eachother. Any read that has identical sequence to any other read isidentified as a duplicate and removed from the population, thus, only asingle copy is retained within the file. Once the duplicate reads havebeen removed, 8 bases are trimmed from each read. The trimmed reads arealigned to a reference genome. Differential expression is thendetermined by comparing FPKM (fragments per kilobase per million reads)values between libraries utilizing scripts such as cufflinks or cuffdiff(Trapnell et al. 2010, Nature Biotechnology, 28, 511-515; Trapnell etal. 2013, Nature Biotechnology, 31, 46-53).

Example 6: Sequencing of the Identifier Site With the Target Sequence inPaired End Reads

The Ovation Library System for Low Complexity Samples (NuGENTechnologies, Inc., San Carlos, Calif.) was used to generate fourlibraries, each from a single amplicon, following manufacturer'sprotocol. Purified libraries were mixed and sequenced as a multiplex onthe Illumina MiSeq (Illumina Inc., San Diego, Calif.) to produce 125ntforward, 8nt index 1, 8nt index 2, and 25nt reverse reads. Because allamplicon reads start and end at the same sequence coordinates, thetraditional method of marking library PCR duplicates (marking reads thatstart and end at the same genomic coordinates as duplicates) cannot beused. Instead, the 0-8nt of random sequence contained in the adaptorsligated to the amplicon were treated as an identifier sequence and usedto mark duplicates. Any paired end reads that shared the same length andsequence of these random bases with any other paired end read was calleda duplicate. The table below shows the results of this duplicatemarking.

TABLE 1 Reads from Duplicate independent Library Reads Reads molecules 1454249 376797 77452 2 439367 364001 75366 3 317760 253057 64703 4 476572398380 78192

Table 1 demonstrates the accuracy of the method used to differentiatebetween duplicate reads and reads from truly independent molecules. Thepopulation of reads from independent molecules, depicted in the finalcolumn of Table 1, represent sequences from independent ampliconmolecules used in generating the libraries.

Example 7: Sequencing of the Identifier Site with the Target Sequence inReduced Representation Bisulfite (RRBS) Libraries

Reduced representation bisulfite (RRBS) libraries of the human genomeare generated through the complete restriction enzyme digestion of 100ng input sample, followed by selection for short fragments. Theresulting pool of fragments are ligated to adaptor sequences comprisingan indexing site and an identifier site. The identifier sites compriseeither 6 or 8 random nucleotides. The sequences are then sequenced toidentify the identifier sites, thus revealing the true number ofduplicates in the pool. In the absence of identifier sites, the numberof apparent or perceived duplicates are greater than the number of trueduplicates. The inclusion of an identifier site results in theidentification of the number of true duplicates, as compared to thelarger number of apparent or perceived duplicates.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as an acknowledgment or anyform of suggestion that they constitute valid prior art or form part ofthe common general knowledge in any country in the world.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1-21. (canceled)
 22. A kit comprising a plurality of syntheticdouble-stranded adaptors, wherein each adaptor comprises: (i) anindexing primer binding site; (ii) an indexing site; (iii) an identifiersite consisting of between 1 and 8 nucleotides; and (iv) a targetsequencing primer binding site; wherein the indexing site is uniqueamongst a subset of the adaptors and is an index for multiplepolynucleotides; and wherein the sequence of the identifier site isvariable in sequence content in a plurality of adaptors.
 23. A kitcomprising a plurality of synthetic double-stranded adaptors, whereineach adaptor comprises: (i) an indexing primer binding site; (ii) anindexing site; (iii) an identifier site consisting of between 1 and 8nucleotides; and (iv) a target sequencing primer binding site; whereinthe indexing site is unique amongst a subset of the adaptors and is anindex for multiple polynucleotides; and wherein the sequence of theidentifier site is not variable in sequence content in a plurality ofadaptors.
 24. The kit of claim 22, wherein the plurality ofdouble-stranded adaptors are divided into two or more containers, andwherein each of the two or more containers contain adaptors sharing thesame indexing site, and no two containers comprise adapters with thesame indexing site.
 25. The kit of claim 22 further comprising one ormore oligonucleotide primers, reagents for amplification, andinstructions for employing the kit.
 26. The kit of claim 25, wherein theone or more oligonucleotide primers and/or reagents for amplificationare contained in single-use aliquots.
 27. The kit of claim 25, whereinthe one or more oligonucleotide primers and/or reagents foramplification are contained in multi-use containers.
 28. The kit ofclaim 22, wherein the adaptors are blunt-ended.
 29. The kit of claim 22,wherein the adaptors are in an aqueous solution.
 30. The kit of claim22, wherein the adaptors are lyophilized.
 31. The kit of claim 22further comprising one or more of the following reagents: a) DNA ligase,b) DNA-dependent DNA polymerase, c) RNA-dependent DNA polymerase, d)forward primer, e) reverse primer, and f) one or more buffers suitablefor one or more of the elements contained in said kit.
 32. The kit ofclaim 22, wherein the adaptors comprise from 5′ to 3′: a) the indexingprimer binding site; b) the indexing site; c) the identifier site; andd) the target sequence primer binding site.
 33. The kit of claim 22,wherein the adaptors comprise from 5′ to 3′: a) the indexing primerbinding site; b) the indexing site; c) the target sequence primerbinding site; and d) the identifier site.
 34. The kit of claim 22,wherein the indexing site is between 2 and 8 nucleotides in length. 35.The kit of claim 22, wherein the indexing site is about 6 nucleotides inlength.
 36. The kit of claim 22, wherein the identifier site is 6nucleotides in length.
 37. The kit of claim 22, wherein the identifiersite is 8 nucleotides in length.
 38. The kit of claim 22, wherein theindexing primer binding site is a universal indexing primer bindingsite.
 39. The kit of claim 22, wherein the target sequence primerbinding site is a universal target sequence primer binding site.
 40. Thekit of claim 22, wherein the identifier site is 5′ to the indexing site.41. The kit of claim 22, wherein the identifier site is heterologous toadjacent sequences.